The primary goal of this proposal is to develop and promote the PI's independent research career by expanding his expertise in molecular genetics, cell culture, and protein expression. These techniques will be added to his current expertise in the biochemistry and biophysics of membranes, and will be applied to investigate the molecular basis of calcium transport regulation in the heart. The research will focus on two membrane proteins found in cardiac sarcoplasmic reticulum (SR)- the calcium pump (CA-ATPase), which pumps Ca into the Sr to relax the muscle, and phospholamban (PLB), which regulates the pump by inhibiting it until PLB is phosphorylated due to stimulation of the cell by adrenaline. The goal of this research is to understand the molecular basis of this process, which is crucial to cardiac performance. The following specific aims will be pursued. 1. Optimize the reconstitution and labeling methods for PLB and Ca- ATPase to perform spectroscopic studies to understand the function, molecular interaction and structural changes in PLB and CA-ATPase. 2. Perturb the structure of PLB using molecular genetics and test the effects of these mutations on structure and function of PLB to understand structural features of PLB essential for its function. 3. Molecular genetics will be used to perturb the structure of Ca-ATPase and study the function, molecular dynamics and physical interactions between Ca-ATPase and PLB or PLB mutants. Molecular genetics and protein expression in tissue culture, is a powerful approach to understand structure-function and regulation (control) mechanisms of biological processes at the molecular level. This project will give the PI an opportunity to master these techniques and combine them with his expertise in biochemical and biophysical methods, producing an ideal combination of investigative tools to develop his independent research career. The success of this project and PI's goal to establish an independent investigator will be aided by outstanding departmental facilities, and by excellent intellectual environment, which include similar research groups with strong expertise in membrane molecular biology and biophysics. The goal of this research program is to understand the molecular basis of cardiac calcium regulation-clearly has profound implications for the development of strategies for treating heart disease.